Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
  • C07D519/04—Dimeric indole alkaloids, e.g. vincaleucoblastine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6805—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a vinca alkaloid
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S424/00—Drug, bio-affecting and body treating compositions
  • Y10S424/809—Drug, bio-affecting and body treating compositions involving immunoglobulin or antibody fragment, e.g. fab', fv, fc, heavy chain or light chain
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/807—Hapten conjugated with peptide or protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/828—Cancer
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/863—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving IgM
  • Y10S530/864—Monoclonal
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/866—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain

Definitions

• the present invention relates to new conjugates of vinca alkaloids of the indole-dihydroindole type with proteins or protein fragments which are endowed with pharmaceutical properties, and especially cytostatic activity.
• Vinca alkaloids of the indole-dihydroindole type in particular vinblastine, vincristine and vindesine, are used in the treatment of cancer.
• the chemotherapeutic use of these derivatives is, however, limited in its effectiveness by their side effects. For this reason, many derivatives have been synthesized in order to reduce these side effects.
• the present invention relates to new products which are conjugates of vinca alkaloids of the indole-dihydroindole type with proteins or protein fragments, wherein the coupling is also accomplished via an ester group derived from the hydroxyl group on carbon 4 of the vinca alkaloid skeleton.
• the subject of the invention consists, more especially, of conjugates of vinca alkaloid of the indoledihydroindole type with a protein or a protein fragment, corresponding to the general formula ##STR2## in which R 1 denotes a protein or a protein fragment;
• R 2 is COO(C 1-3 alkyl) or CO--R 7 where R 7 is NH 2 or an amino acid ester or peptide ester;
• R 3 is H, CH 3 or CHO
• R 6 is H and one of R 4 and R 5 is ethyl and the other is H or OH;
• R 5 and R 6 when R 5 and R 6 are taken together with the carbon atoms to which they are attached, they form an oxirane ring and R 4 is ethyl, and
• A is a residue of a bifunctional organic derivative of the maleoylamino acid or maleoyl peptide or maleoylphenoxy type.
• the protein or protein fragment is coupled to the vinca compound via an arm, by prior condensation of the 4-deactylindole-dihydroindole vinca alkaloid with a bifunctional organic derivative of the maleoylamino acid type or maleoyl peptide type of general formula ##STR3##
• X can also denote a phenyl radical.
• bifunctional derivative defined above comprises asymmetric centers, it can be used in its racemic form or in one of its optically active forms.
• conjugates of the invention can be represented by the following formula: ##STR5## in which R 1 denotes a protein or a protein fragment,
• R 2 is COO(C 1-3 alkyl), or CO--R 7 where R 7 is NH 2 or an aminoacid ester or peptide ester,
• R 3 is H, CH 3 or CHO
• R 6 is H and one of R 4 and R 5 is ethyl and the other is H or OH, and
• R 5 and R 6 when R 5 and R 6 are taken together with the carbon atoms to which they are attached, they form an oxirane ring and R 4 is ethyl.
• R 2 is CO--R 7 , R 7 being an amino acid ester or peptide ester, and R 3 , R 4 , R 5 and R 6 have the meaning stated for the vinblastine derivatives,
• vincristine derivatives where R 2 is COOCH 3 , R 3 is formyl, R 4 is hydroxyl, R 5 is ethyl and R 6 is hydrogen,
• leurosidine derivatives where R 2 is COOCH 3 , R 3 is methyl, R 4 is ethyl, R 5 is hydroxyl and R 6 is hydrogen,
• leurosine derivatives where R 2 is COOCH 3 , R 3 is methyl, R 4 is ethyl, and R 5 and R 6 together form an epoxide link.
• the bifunctional derivative of the maleoyl peptide type can also result from the condensation of an N-alkoxymaleimide with a dipeptide or tripeptide, to give a derivative --(X 1 --NH--CO--) n X 1 where X 1 can have the same meaning as X described above.
• proteins which can advantageously be used are, in particular, bovine or human serum albumin, or fetuin or immunoglobulins.
• the proteins used can also be treated in order to be selectively modified. These modifications enable protein conjugates to be obtained which, when they are used therapeutically, will be preferentially concentrated in certain tissues, for example in the liver. It is thus possible, prior to the condensation of the vinca alkaloid derivative with the protein, to galactosylate the latter.
• the immunoglobulins specific for malignant cell surface antigens, and the techniques for producing them from serum of immunized animals or by culturing hybridomas which secrete monoclonal antibodies, are well known.
• the preferred type of antibody for use in the invention is an immunoglobulin of the IgG class of human origin.
• immunoglobulins of other species are also included in this invention.
• Some representative immunoglobulins are as follows:
• monoclonal Ig from mouse hybridomas secreting antibodies which react with human ovarian carcinoma cells
• p1 monoclonal Ig from mouse hybridomas secreting antibodies which react with human osteosarcoma cells and
• the conjugates can also be prepared with immunoglobulin fragments, namely Fab, Fab' or F(ab') 2 fragments or monomeric IgM, obtained from an antibody by digestion with a proteolytic enzyme.
• the conjugates of the present invention are obtained, in a first stage, by esterification of a maleoylamino acid or a maleoyl peptide with the C 4 -hydroxyl of a 4-deacetylindole-dihydroindole vinca alkaloid of formula II, to give a derivative of formula III, ##STR7## in which X, R 2 , R 3 , R 4 , R 5 and R 6 have the meaning stated above.
• the 4-carboxy-maleoyl vinca derivatives III are then condensed with a protein or a protein fragment by addition of either the free thiol groups or the free amino groups of the protein, for example the amino groups derived from lysine residues of the protein, to the olefinic double bond of the maleimide, according to a Michael type addition mechanism (Means, G. E. and Feeney, R. E.; Chemical modification of proteins, 1971, p. 110-138, Holden Day Inc., San Francisco) to give the conjugates I.
• Michael type addition mechanism Means, G. E. and Feeney, R. E.; Chemical modification of proteins, 1971, p. 110-138, Holden Day Inc., San Francisco
• maleoylamino acids or maleoyl peptides are produced according to the methods described by O. Keller and J. Rudinger, Helv. 58, 531 (1975), by D. H. Rich, P. D. Gesselchen, A. Tong, A. Cheung and C. K. Buckner, J. Med. Chem, 18, 1004 (1975) and by A. Sato and M. Nakao, J. Biochem., 90, 1117 (1981).
• the condensation of the maleoyl derivatives with the vinca alkaloid can be performed in the conventional manner with an alkyl chloroformate, preferably ethyl or isobutyl chloroformate, in the presence of an amine base such as triethylamine, N-methylpiperidine or N-methylmorpholine, in an organic solvent such as ethyl acetate, tetrahydrofuran or methylene chloride.
• an alkyl chloroformate preferably ethyl or isobutyl chloroformate
• an amine base such as triethylamine, N-methylpiperidine or N-methylmorpholine
• organic solvent such as ethyl acetate, tetrahydrofuran or methylene chloride.
• the derivative obtained is isolated from the reaction medium and purified by means of classical methods used in chemistry.
• the production of the conjugates can be carried out by reacting the protein, the polyclonal or monoclonal antibody, with the vinca compound of formula III under classical conditions, for example in aqueous medium at a temperature of between 4° C. and 40° C. and a pH of 7.5 to 9.5.
• the numbers of residues attached can depend on the concentration of the reagents and the reaction time, but the average number is generally between 5 and 20.
• a solution of the compound III in an organic solvent such as dioxane is added dropwise to a buffered solution of protein, for example in 0.1M phosphate buffer at pH 8.2. After the mixture is left overnight at room temperature, the conjugate is isolated by gel filtration, concentrated by ultrafiltration and sterilized. The protein content is measured by the Lowry method and the alkaloid content estimated by measuring the radioactivity.
• the maleoyl link makes it possible to conjugate both the free amino groups and thiol groups of the protein or protein fragment.
• digestion with lysosomal enzymes indicates that the vinca alkaloid is liberated to a much greater extent in the case of the maleoyl link.
• the conjugates are also stable in serum and at acid pH.
• the compounds of the invention were tested on BDF 1 mice in which a P388 leukemia had been implanted intraperitoneally. The first results indicate that the compounds show significant activity on this experimental model since they induce an increase in the survival time.
• the new conjugates of the invention show antitumor properties which are especially advantageous and capable of being used in human therapy.
• the compounds of the invention are preferably administered parenterally, dissolved in a pharmaceutically acceptable solvent.
• a pharmaceutically acceptable solvent Physiological saline or other solutions buffered, for example, with a phosphate are suitable solvents.
• the active substance is generally administered at a dosage which can vary from 50 mg to several grams.
• the compounds of the invention can, in addition, be used in combination with other antitumor agents.
• the solution is diluted with 200 ml of water and stirred at 40° C. for 1 hour.
• the pH of the solution (8.2) is then brought to 6.4 by adding concentrated surfuric acid.
• the solution is then concentrated to 100 ml and acidified to pH 2 by adding 1M sulfuric acid, and is extracted three times with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate and evaporated to dryness under vacuum.
• IR spectrum (KBr) cm -1 3400, 3100, 2930, 1780, 1740, 1700, 1460, 1420, 1390, 1370, 1345, 1220, 1175, 1118, 1080, 1068, 1015, 970, 835, 700.
• a solution of 4.2 g (32.3 mmol) of 6-aminocaproic acid in 163 ml of saturated sodium bicarbonate solution is treated at 0° C., with vigorous stirring, with 5 g (32.3 mmol) of N-carbomethoxymaleimide. After 1 hour, the solution is diluted with 256 ml of water and stirred at room temperature for 1 hour. The pH of the solution (8.2) is then brought to 6.4 by adding concentrated sulfuric acid. The solution is then concentrated to 100 ml and acidified to pH 2 by adding 1M sulfuric acid, and is extracted three times with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate and evaporated to dryness under vacuum. The residue is purified by chromatography on a silica column (elution: chloroform/acetic acid, 95:5). In this manner, 4.7 g of N-maleoyl-6-aminocaproic acid are obtained.
• IR spectrum (KBr/cm -1 ): 3,090; 2,940; 2,880; 1,770; 1,710; 1,470; 1,450; 1,380; 1,370; 1,340; 1,315; 1,260; 1,210; 1,135; 1,110; 1,140; 1,100; 1,015; 1,000; 815; 840; 735; 700.
• N-maleoyl-L-isoleucine is obtained by treating 424 mg (3.22 mol) of L-isoleucine with 500 mg (3.2 mmol) of N-carbomethoxymaleimide.
• Mass spectrum (CDI, isobutane): 423 (2M+1), 352, 270, 212 (M + +1), 188, 166, 132, 123.
• the solution is diluted with 91 ml of water and stirred at 40° C. for one hour.
• the pH of the solution (8.2) is then brought to 6.4 by adding concentrated sulfuric acid.
• the solution is then concentrated to 50 ml and acidified to pH 2 by adding 1M sulfuric acid, and is extracted three times with ethyl acetate.
• the residue obtained is purified by chromatography on a silica column (elution: chloroform/acetic acid, 95:5). In this manner, 267 mg of pure product are obtained.
• a solution of maleic anhydride (2.4 g; 24.8 mmol) in 10.1 ml of acetic acid is added to a solution of 11-aminoundecanoic acid (5 g; 24.8 mmol) in 30 ml of acetic acid, and the mixture is maintained with vigorous stirring at room temperature for 3 hours.
• the white precipitate is filtered off, washed with cold acetic acid and dried (6.3 g; 21.1 mmol; 85%). 3 g of the precipitate (10.00 mmol) are dissolved in dry toluene (300 ml) and treated with triethylamine (2 g; 22.2 mmol).
• IR spectrum (KBr, cm -1 ): 3450, 3100, 2910, 2850, 1770, 1700, 1610, 1590, 1470, 1450, 1420, 1375, 1340, 1310, 1280, 1240, 1180, 1125, 840, 700.
• a solution of maleic anhydride (2.28 g; 23.22 mmol) in 9.6 ml of acetic acid is added to a solution of 12-aminododecanoic acid (5 g; 23.22 mmol) in 28 ml of acetic acid, and the mixture is maintained with vigorous stirring at room temperature for 3 hours.
• IR spectrum (KBr, cm -1 ): 3450, 3080, 2920, 2825, 1770, 1470, 1450, 1440, 1415, 1380, 1340, 1300, 1260, 1250, 1205, 1120, 920, 840, 700.
• a solution of 1.17 ml (0.009 mol) of isobutyl chloroformate in 5 ml of ethyl acetate is added dropwise to a solution of 1.52 g (0.009 mol) of N-maleoylalanine and 1.25 ml (0.009 mol) of triethylamine in 10 ml of ethyl acetate cooled to 0° C.
• the mixture is stirred for 1 h 30 min at 0° C., and 2.3 g (0.003 mol) of 0 4 -deacetylvinblastine dissolved in 20 ml of ethyl acetate are added while the temperature is kept at 0° C.
• the mixture is then allowed to return to room temperature and is stirred for 10 hours.
• 50 ml of ethyl acetate and 50 ml of 10% strength aqueous sodium carbonate solution are added.
• the mixture is stirred and the organic phase decanted and separated.
• the aqueous phase is extracted three times with ethyl acetate.
• the combined organic phases are washed with an aqueous solution, dried over magnesium sulfate and evaporated to dryness under vacuum.
• the residue obtained is purified by chromatography on a silica column (elution: dichloromethane/methanol, 92:8). 1.86 g of pure product is thereby obtained.
• Mass spectrum (DCI, isobutane): 935 (M+14), 922 (M+1), 921 (M), 751, 693, 519, 445, 371, 133.
• a solution of 371 ⁇ l (2.861 mmol) of isobutyl chloroformate in 1 ml of ethyl acetate is added dropwise to a solution of 604 mg (2.861 mmol) of N-maleoyl-6-aminocaproic acid and 514 ⁇ l (4.65 mmol) of N-methylmorpholine in 4 ml of ethyl acetate cooled to 0° C.
• the mixture is stirred for 3 minutes at 0° C., and 550 mg (0.715 mmol) of 0 4 -deacetylvinblastine dissolved in 1 ml of ethyl acetate is then added while the temperature is kept at 0° C.
• Mass spectrum (DCI, isobutane): 993; 979; 965 (M + +4); 963 (M + +2); 961 (M + ); 946; 933; 920; 906; 812; 754; 693.
• IR spectrum (KBr, cm -1 ): 3,400; 3,050; 2,940; 1,740; 1,700; 1,615; 1,500; 1,460; 1,440; 1,410; 1,370; 1,220; 1,170; 1,040.
• a solution of 152 ⁇ l (1.170 mmol) of isobutyl chloroformate in 1 ml of ethyl acetate is added dropwise to a solution of 282 mg (1.170 mmol) of N-maleoyl-L-glutamic acid ⁇ -methyl ester and 163 ⁇ l (1.170 mmol) of triethylamine in 1.4 ml of ethyl acetate cooled to 0° C.
• the mixture is stirred for 4 min at 0° C., and 300 mg (0.390 mmol) of 0 4 -deactylvinblastine dissolved in 1 ml of ethyl acetate are then added while the temperature is kept at 0° C.
• a solution of 92 ⁇ l (0.7109 mmol) of isobutyl chloroformate in 1 ml of ethyl acetate is added dropwise to a solution of 150 mg (0.7109 mmol) of N-maleoyl-L-isoleucine and 99 ⁇ l (0.7109 mmol) of triethylamine in 1 ml of ethyl acetate cooled to 0° C.
• the mixture is stirred for 3 min at 0° C., and 182 mg (0.237 mmol) of 0 4 -deacetylvinblastine dissolved in 1 ml of ethyl acetate is then added while the temperature is kept at 0° C.
• IR spectrum (KBr, cm -1 ): 3,480-3,400; 2,960; 2,920; 2,880; 1,775; 1,740; 1,710; 1,610; 1,500; 1,460; 1,430; 1,380; 1,250; 1,200; 1,040; 1,000; 830; 740.
• IR spectrum (KBr, cm -1 ) 3460, 3030, 2960, 2880, 1770, 1740, 1715, 1610, 1500, 1460, 1430, 1410, 1380, 1260, 1220, 1175, 1110, 1025, 910, 800, 730, 700.
• IR spectrum (KBr, cm -1 ) 3430, 3040, 2930, 2860, 1770, 1735, 1710, 1615, 1505, 1460, 1410, 1370, 1230-1250, 1000-1100.
• Mass spectrum (DCI, isobutane): 1089, 1064, 1048, 1034, 1000, 990, 976, 960.
• IR spectrum (KBr, cm -1 ): 3460, 3040, 2930, 2860, 1735, 1700, 1610, 1500, 1460, 1430, 1410, 1365, 1245, 1220.
• a solution of 150 mg (0.163 mmol) of 4-(N-maleoyl-L-alanyl)vinblastine in 1 ml of ethanol is added dropwise to a solution of 40.26 mg (0.163 mmol) of lysine ethyl ester HCl and 150 ⁇ l (0.978 mmol) of triethylamine in 7.5 ml of absolute ethanol.
• the mixture is stirred overnight at room temperature and the solution then evaporated.
• the residue obtained is purified by chromatography on a silica column (elution: 14% MeOH/NH 3 in ether). 101.2 mg of pure product are thereby obtained.
• Mass spectrum (DCI, isobutane): 1,095 (M + +2); 1,094 (M + +1); 1,037; 921; 839; 769; 693; 615; 574; 532.
• the protein content is measured by Lowry method and the alkaloid content is estimated by measuring the radioactivity.
• the conjugate obtained contains 13.5 mols of alkaloid per mol of galactosylated human albumin.
• a solution of 591 mg of HAgal in 16.5 ml of 0.4M phosphate buffer pH 8.5 is prepared separately.
• the protein content is measured by the Lowry method and the alkaloid content is estimated by measuring the radioactivity.
• the conjugate obtained contains 8.8 mols of alkaloid per mol of galactosylated human albumin.
• the protein content is measured by the Lowry method and the alkaloid content estimated by measuring the radioactivity.
• the conjugate obtained contains 5.7 mols of alkaloid per mol of galactosylated human albumin.
• a solution of 969 mg of HAgal in 27 ml of 0.4M phosphate buffer pH 8.5 is prepared separately.
• the protein content is measured by the Lowry method and the alkaloid content estimated by measuring the radioactivity.
• the conjugate obtained contains 10.2 mols of alkaloid per mol of galactosylated human albumin.
• the protein content is measured by the Lowry method and the alkaloid content estimated by measuring the radioactivity.
• the conjugate obtained contains 9 mols of alkaloid per mol of galactosylated human albumin.
• the excluded peak is collected and the quantity is determined.
• the protein content is measured by the Lowry Method and the alcaloid content is estimated by determination of the radioactivity.
• the obtained conjugate contains 6.6 moles alcaloid per mole immunoglobuline.
• the compounds of the invention were subjected to a pharmacological study.
• the sensitivity of the conjugates to lysosomal enzymes was studied by incubation of these conjugates for 48 hours at 37° C. in the presence of 5 mM cystein, 40 mM acetate buffer and lysosomal enzymes.
• the radioactivity of the supernatant is estimated by liquid scintillation counting of an aliquot portion.
• the soluble radioactivity is a measure of the digestion of the conjugate.
• V 4 -C 6 -Mal-HAg 100%
• the stability of the conjugates in the presence of serum was studied by incubating the conjugate in the presence of 62% fetal calf serum at 37° C. for 48 hours.
• the undegraded proteins are precipitated by adding one volume of trichloroacetic acid (40% strength TCA). After incubation of the samples at 4° C. for one hour, the latter are centrifuged and the radioactivity of the supernatant is estimated by liquid scintillation counting of an aliquot portion.
• trichloroacetic acid 50% strength TCA
• the soluble radioactivity is a measure of the digestion of the conjugate.
• the stability of the conjugates at acid pH was studied by incubating the conjugate in the presence of 40 mM acetate buffer, pH 4.5, at 37° C. for 48 hours.
• the digestion is estimated by the TCA precipitation technique.
• the chemotherapeutic activity of the intermediate derivatives and the conjugates was assessed on leukemia P388 administered i.p. to female BDF 1 mice: 10 6 tumor cells are inoculated i.p. on day 0. The conjugate is administered i.p. on day 1.
• the ILS value represents the percentage survival time of the treated mice compared with that of untreated mice.
• the mol ratio indicates the number of mols of alkaloid per mol of galactosylated human albumin.
• mice surviving at day 60 The number of mice surviving at day 60 is shown, as well as the dose in mg/kg of the alkaloid and the protein.

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• 1986
  • 1986-12-03 EP EP86870179A patent/EP0232693A3/de not_active Withdrawn
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